The forage legume alfalfa (Medicago sativa) has high nutritional value and low fiber content and, consequently, is an important dairy feed. Traits such as these, as well as others, can be improved upon by conventional plant transformation techniques. In this regard, conventional methods of transforming alfalfa typically rely on infecting alfalfa leaf explants from highly regenerable clones to produce transformed alfalfa plants (Bingham, Crop Sci., 31:1098, 1991; Austin, et al., Euphytica, 85: 381-393, 1995). These methods, however, require multiple manipulative steps, such selection, somatic embryogenesis, and regeneration, which prove labor-intensive, time consuming, and costly.
Moreover, conventional transformation methods typically introduce foreign DNA into the alfalfa genome permanently. Such foreign DNA generally includes selectable marker genes, extended Agrobacterium T-DNA border regions, and regulatory elements from viral and bacterial origins. The presence of foreign DNA in foodstuffs destined for human consumption may cause concern to some. For this reason, Rommens et al. in U.S. patent publication serial No. 2003/0221213, were the first to propose the all native approach to genetic engineering. In this approach, only DNA from the genome of the organism of interest, or DNA accessible to the organism of interest through naturally breeding, is introduced via genetic engineering techniques. Plants obtained through this new plant breeding process do not contain foreign nucleic acid but only contain nucleic acid from the plant species selected for transformation, or plants that are sexually compatible with the selected plant species. Potato plants (Solanum tuberosum) that display enhanced black spot bruise tolerance represent plants generated through genetic engineering with only native DNA obtained from the potato genome. The tools that were developed for this purpose include efficient marker-free transformation and replacement of foreign genetic elements involved in gene transfer and expression by DNA sequences from the target plant species.
Interestingly, various important improvements for alfalfa may be accomplished by modifying the expression of endogenous genes and would, thus, not require foreign genes. For instance, it has been shown that reduced expression of endogenous caffeic acid o-methyltransferase (Comt) or caffeoyl CoA 3-O-methyltransferase (Ccomt) genes triggered up to 30% decreases in lignin content and consequently enhanced the nutritional value of alfalfa forage (Guo et al., Plant Cell 13: 73-88, 2001; Guo et al., Transgenic Res 10: 457-464, 2001), at the same time also reducing the amount of waste manure by-product. The availability of all-native DNA transformation methods would make it possible to improve the alfalfa crop while addressing key public concerns. The development of such methods is described herein, as is the exploitation of these methods to reduce lignin content in intragenic alfalfa plants.